Tuberculosis (TB) is the leading cause of adult death by a single infectious organism, accounting for over 2.3 million deaths per year worldwide. The emergence of drug resistance and the increased spread of TB promoted by the global epidemic of HIV are worsening the global tuberculosis problem. Clearly, novel strategies are needed to control the tuberculosis epidemic. The acquisition of basic knowledge of the causative agent of TB, Mycobacterium tuberculosis, is a prerequisite for developing such tools. The goal of this proposal has been to develop the set of tools necessary for the generation of mutants and the transfer of genes M. tuberculosis mutants. Using mycobacteriophages as a starting point, we have developed the first transformation systems for mycobacteria, the ability to generate random mutants with transposons, and, recently, the ability to efficiently generate targeted gene disruptions using specialized transduction. The combination of these tools with the acquisition of the genomic sequence of M. tuberculosis provides unprecedented opportunities to acquire basic knowledge of the tubercle bacillus. Indeed, our successes in developing these tools have allowed us to: i) determine the previously unknown targets of the anti-TB drugs isoniazid and ethionamide, ii) identify numerous novel genes required for M. tuberculosis virulence, and iii) to determine the genetic and functional basis for the attenuation of the TB vaccine strain, BCG. This proposal seeks to use these genetic systems in combinations with a number of newly developed screens for mutants defective in virulence traits. The tubercle bacillus has acquired the means to invade and multiply in macrophages. Moreover, M. tuberculosis has evolved mechanisms by which it overcomes effector mechanisms of the host's innate and adaptive immune responses. We plan to elucidate the mechanisms by which M. tuberculosis causes pathogenesis and overcomes the innate and adaptive immune responses of the host by screening for mutants that are defective in their abilities to: i) grow in mammalian lungs, ii) to modulate different cytokine responses, and iii) to grow and persist in immunocompetent and immunocompromised mice. Ultimately, the knowledge of mycobacterial virulence and persistence factors should lead to the development of novel drugs, vaccines, and immunotherapies to control tuberculosis. [unreadable] [unreadable]